Long-term structural brain changes in adult rats after mild ischaemic stroke.

Preclinical studies of remote degeneration have largely focused on brain changes over the first few days or weeks after stroke. Accumulating evidence suggests that neurodegeneration occurs in other brain regions remote to the site of infarction for months and even years following ischaemic stroke. Brain atrophy appears to be driven by both axonal degeneration and widespread brain inflammation. The evolution and duration of these changes are increasingly being described in human studies, using advanced brain imaging techniques. Here, we sought to investigate long-term structural brain changes in a model of mild focal ischaemic stroke following injection of endothlin-1 in adult Long-Evans rats (n = 14) compared with sham animals (n = 10), over a clinically relevant time-frame of 48 weeks. Serial structural and diffusion-weighted MRI data were used to assess dynamic volume and white matter trajectories. We observed dynamic regional brain volume changes over the 48 weeks, reflecting both normal changes with age in sham animals and neurodegeneration in regions connected to the infarct following ischaemia. Ipsilesional cortical volume loss peaked at 24 weeks but was less prominent at 36 and 48 weeks. We found significantly reduced fractional anisotropy in both ipsi- and contralesional motor cortex and cingulum bundle regions of infarcted rats (P < 0.05) from 4 to 36 weeks, suggesting ongoing white matter degeneration in tracts connected to but distant from the stroke. We conclude that there is evidence of significant cortical atrophy and white matter degeneration up to 48 weeks following infarct, consistent with enduring, pervasive stroke-related degeneration.

Effects of wheel-running on anxiety and depression-relevant behaviours in the MCAO mouse model of stroke: moderation of brain-derived neurotrophic factor and serotonin receptor gene expression.

Stroke continues to be a major cause of mortality globally. Post-stroke treatment is complicated by the heterogenous nature of pathology and the emergence of secondary psychological symptoms are an additional challenge to the recovery process. Poststroke depression (PSD) is a common co-morbidity and is a major impediment to recovery. While selective serotonin reuptake inhibitors (SSRIs) have proven to be clinically efficacious in treating PSD, the pathogenic processes that underlie the manifestation of depressive mood post-stroke remains unclear. Furthermore, the use of SSRIs is associated with risks of intracerebral haemorrhage, so alternative treatment options need to be continuously explored. Exercise has been demonstrated to be beneficial for improving mood in humans and preclinical models of neurological conditions. Little is known of the mood-related benefits of physical exercise post-stroke. Using the middle cerebral artery occlusion (MCAO) mouse model of cerebral ischaemia, we investigated whether behavioural deficits emerge post-MCAO and could be rescued by voluntary wheel-running. We report that MCAO induced hypo-locomotion and anhedonia-related behaviours, with some improvements conferred by wheel-running. Serotonin transporter gene expression was increased in the MCAO hippocampus and frontal cortex, but this increase remained despite wheel-running. Wheel-running associated up-regulation of BDNF gene expression was unaffected in MCAO mice, reflecting conservation of key neuroplasticity molecular pathways. Taken together, our results highlight the need for further research into serotonergic modulation of the affective symptoms of stroke.

Hemispheric cortical atrophy and chronic microglial activation following mild focal ischemic stroke in adult male rats.

Animal modeling has played an important role in our understanding of the pathobiology of stroke. The vast majority of this research has focused on the acute phase following severe forms of stroke that result in clear behavioral deficits. Human stroke, however, can vary widely in severity and clinical outcome. There is a rapidly building body of work suggesting that milder ischemic insults can precipitate functional impairment, including cognitive decline, that continues through the chronic phase after injury. Here we show that a small infarction localized to the frontal motor cortex of rats following injection of endothelin-1 results in an essentially asymptomatic state based on motor and cognitive testing, and yet produces significant histopathological change including remote atrophy and inflammation that persists up to 1 year. While there is understandably a major focus in stroke research on mitigating the acute consequences of primary infarction, these results point to progressive atrophy and chronic inflammation as additional targets for intervention in the chronic phase after injury. The present rodent model provides an important platform for further work in this area.

Longitudinal hippocampal volumetric changes in mice following brain infarction.

Hippocampal atrophy is increasingly described in many neurodegenerative syndromes in humans, including stroke and vascular cognitive impairment. However, the progression of brain volume changes after stroke in rodent models is poorly characterized. We aimed to monitor hippocampal atrophy occurring in mice up to 48-weeks post-stroke. Male C57BL/6J mice were subjected to an intraluminal filament-induced middle cerebral artery occlusion (MCAO). At baseline, 3-days, and 1-, 4-, 12-, 24-, 36- and 48-weeks post-surgery, we measured sensorimotor behavior and hippocampal volumes from T2-weighted MRI scans. Hippocampal volume-both ipsilateral and contralateral-increased over the life-span of sham-operated mice. In MCAO-subjected mice, different trajectories of ipsilateral hippocampal volume change were observed dependent on whether the hippocampus contained direct infarction, with a decrease in directly infarcted tissue and an increase in non-infarcted tissue. To further investigate these volume changes, neuronal and glial cell densities were assessed in histological brain sections from the subset of MCAO mice lacking hippocampal infarction. Our findings demonstrate previously uncharacterized changes in hippocampal volume and potentially brain parenchymal cell density up to 48-weeks in both sham- and MCAO-operated mice.

Dendritic Cells and Microglia Have Non-redundant Functions in the Inflamed Brain with Protective Effects of Type 1 cDCs.

Brain CD11c+ cells share features with microglia and dendritic cells (DCs). Sterile inflammation increases brain CD11c+ cells, but their phenotype, origin, and functions remain largely unknown. We report that, after cerebral ischemia, microglia attract DCs to the inflamed brain, and astroglia produce Flt3 ligand, supporting development and expansion of CD11c+ cells. CD11c+ cells in the inflamed brain are a complex population derived from proliferating microglia and infiltrating DCs, including a major subset of OX40L+ conventional cDC2, and also cDC1, plasmacytoid, and monocyte-derived DCs. Despite sharing certain morphological features and markers, CD11c+ microglia and DCs display differential expression of pattern recognition receptors and chemokine receptors. DCs excel CD11c- and CD11c+ microglia in the capacity to present antigen through MHCI and MHCII. Of note, cDC1s protect from brain injury after ischemia. We thus reveal aspects of the dynamics and functions of brain DCs in the regulation of inflammation and immunity.

Role of the S1P pathway and inhibition by fingolimod in preventing hemorrhagic transformation after stroke.

Hemorrhagic transformation (HT) is a complication of severe ischemic stroke after revascularization. Patients with low platelet counts do not receive reperfusion therapies due to high risk of HT. The immunomodulatory drug fingolimod attenuated HT after tissue plasminogen activator in a thromboembolic stroke model, but the underlying mechanism is unknown. Fingolimod acts on several sphingosine-1-phosphate (S1P) receptors, prevents lymphocyte trafficking to inflamed tissues, and affects brain and vascular cells. This study aimed to investigate changes in S1P-signaling in response to brain ischemia/reperfusion and the effects of the S1P receptor modulator fingolimod on HT. We studied brain expression of S1P signaling components, S1P concentration, and immune cell infiltration after ischemia/reperfusion in mice. We administered fingolimod after ischemia to wild-type mice, lymphocyte-deficient Rag2-/- mice, and mice with low platelet counts. Ischemia increased S1P-generating enzyme SphK1 mRNA, S1P concentration, and S1P receptor-1 (S1P1)+ T-cells in the brain. Fingolimod prevented lymphocyte infiltration, and attenuated the severity of HT in Rag2-/- mice but it was ineffective under thrombocytopenia. Fingolimod prevented ß-catenin degradation but not Evans blue extravasation. Ischemia/reperfusion upregulates brain S1P signaling pathway, and fingolimod exerts local effects that attenuate HT. Although fingolimod seems to act on the brain tissue, it did not prevent blood-brain barrier leakage.

CD69 Plays a Beneficial Role in Ischemic Stroke by Dampening Endothelial Activation.

RATIONALE: CD69 is an immunomodulatory molecule induced during lymphocyte activation. Following stroke, T-lymphocytes upregulate CD69 but its function is unknown. OBJECTIVE: We investigated whether CD69 was involved in brain damage following an ischemic stroke. METHODS AND RESULTS: We used adult male mice on the C57BL/6 or BALB/c backgrounds, including wild-type mice and CD69-/- mice, and CD69+/+ and CD69-/- lymphocyte-deficient Rag2-/- mice, and generated chimeric mice. We induced ischemia by transient or permanent middle cerebral artery occlusion. We measured infarct volume, assessed neurological function, and studied CD69 expression, as well as platelet function, fibrin(ogen) deposition, and VWF (von Willebrand factor) expression in brain vessels and VWF content and activity in plasma, and performed the tail-vein bleeding test and the carotid artery ferric chloride-induced thrombosis model. We also performed primary glial cell cultures and sorted brain CD45-CD11b-CD31+ endothelial cells for mRNA expression studies. We blocked VWF by intravenous administration of anti-VWF antibodies. CD69-/- mice showed larger infarct volumes and worse neurological deficits than the wild-type mice after ischemia. This worsening effect was not attributable to lymphocytes or other hematopoietic cells. CD69 deficiency lowered the time to thrombosis in the carotid artery despite platelet function not being affected. Ischemia upregulated Cd69 mRNA expression in brain endothelial cells. CD69-deficiency increased fibrin(ogen) accumulation in the ischemic tissue, and plasma VWF content and activity, and VWF expression in brain vessels. Blocking VWF reduced infarct volume and reverted the detrimental effect of CD69-/- deficiency. CONCLUSIONS: CD69 deficiency promotes a prothrombotic phenotype characterized by increased VWF and worse brain damage after ischemic stroke. The results suggest that CD69 acts as a downregulator of endothelial activation.

T Cells Prevent Hemorrhagic Transformation in Ischemic Stroke by P-Selectin Binding.

Objective- Hemorrhagic transformation is a serious complication of ischemic stroke after recanalization therapies. This study aims to identify mechanisms underlying hemorrhagic transformation after cerebral ischemia/reperfusion. Approach and Results- We used wild-type mice and Selplg-/- and Fut7-/- mice defective in P-selectin binding and lymphopenic Rag2-/- mice. We induced 30-minute or 45-minute ischemia by intraluminal occlusion of the middle cerebral artery and assessed hemorrhagic transformation at 48 hours with a hemorrhage grading score, histological means, brain hemoglobin content, or magnetic resonance imaging. We depleted platelets and adoptively transferred T cells of the different genotypes to lymphopenic mice. Interactions of T cells with platelets in blood were studied by flow cytometry and image stream technology. We show that platelet depletion increased the bleeding risk only after large infarcts. Lymphopenia predisposed to hemorrhagic transformation after severe stroke, and adoptive transfer of T cells prevented hemorrhagic transformation in lymphopenic mice. CD4+ memory T cells were the subset of T cells binding P-selectin and platelets through functional P-selectin glycoprotein ligand-1. Mice defective in P-selectin binding had a higher hemorrhagic score than wild-type mice. Adoptive transfer of T cells defective in P-selectin binding into lymphopenic mice did not prevent hemorrhagic transformation. Conclusions- The study identifies lymphopenia as a previously unrecognized risk factor for secondary hemorrhagic transformation in mice after severe ischemic stroke. T cells prevent hemorrhagic transformation by their capacity to bind platelets through P-selectin. The results highlight the role of T cells in bridging immunity and hemostasis in ischemic stroke.

Acute or Delayed Systemic Administration of Human Amnion Epithelial Cells Improves Outcomes in Experimental Stroke.

BACKGROUND AND PURPOSE: Human amnion epithelial cells (hAECs) are nonimmunogenic, nontumorigenic, anti-inflammatory cells normally discarded with placental tissue. We reasoned that their profile of biological features, wide availability, and the lack of ethical barriers to their use could make these cells useful as a therapy in ischemic stroke. METHODS: We tested the efficacy of acute (1.5 hours) or delayed (1-3 days) poststroke intravenous injection of hAECs in 4 established animal models of cerebral ischemia. Animals included young (7-14 weeks) and aged mice (20-22 months) of both sexes, as well as adult marmosets of either sex. RESULTS: We found that hAECs administered 1.5 hours after stroke in mice migrated to the ischemic brain via a CXC chemokine receptor type 4-dependent mechanism and reduced brain inflammation, infarct development, and functional deficits. Furthermore, if hAECs administration was delayed until 1 or 3 days poststroke, long-term functional recovery was still augmented in young and aged mice of both sexes. We also showed proof-of-principle evidence in marmosets that acute intravenous injection of hAECs prevented infarct development from day 1 to day 10 after stroke. CONCLUSIONS: Systemic poststroke administration of hAECs elicits marked neuroprotection and facilitates mechanisms of repair and recovery.

Selective Sphingosine 1-Phosphate Receptor 1 Agonist Is Protective Against Ischemia/Reperfusion in Mice.

BACKGROUND AND PURPOSE: Growing evidence supports that the immunomodulatory drug fingolimod is protective in stroke. Fingolimod binds to 4 of 5 sphingosine-1-phosphate (S1P) receptors and, among other actions, it induces lymphopenia. In this study, we investigated whether a selective S1P1 agonist is protective in experimental stroke. METHODS: Drug selectivity was studied in vitro in cells overexpressing the human S1P receptors. Mice (n=54) received different doses of LASW1238 (3 or 10 mg/kg), fingolimod (1 mg/kg), or the vehicle intraperitoneal, and lymphopenia was studied at different time points. After intraluminal middle cerebral artery occlusion for 45 minutes and immediately after reperfusion, mice (n=56) received the drug treatment. At 24 hours, a neurological test was performed and infarct volume was measured. Treatment and all the analyses were performed in a blind fashion. RESULTS: In vitro functional assays showed that LASW1238 is a selective agonist of the S1P1 receptor. At 10 mg/kg, this compound induced sustained lymphopenia in mice comparable with fingolimod, whereas at 3 mg/kg it induced short-lasting lymphopenia. After ischemia, both LASW1238 (10 mg/kg) and fingolimod reduced infarct volume, but only LASW1238 (10 mg/kg) showed statistically significant differences versus the vehicle. The neurological function and plasma cytokine levels were not different between groups. CONCLUSIONS: The selective S1P1 agonist LASW1238 reduces infarct volume after ischemia/reperfusion in mice, but only when lymphopenia is sustained for at least 24 hours. S1P1 and lymphocytes are potential targets for drug treatment in stroke. Defining the best drug dosing regimens to control the extent and duration of lymphopenia is critical to achieve the desired effects.

Sex-dependent effects of G protein-coupled estrogen receptor activity on outcome after ischemic stroke.

BACKGROUND AND PURPOSE: Experimental studies indicate that estrogen typically, but not universally, has a neuroprotective effect in stroke. Ischemic stroke increases membrane-bound G protein-coupled estrogen receptor (GPER) distribution and expression in the brain of male but not female mice. We hypothesized that GPER activation may have a greater neuroprotective effect in males than in females after stroke. METHODS: Vehicle (dimethyl sulfoxide), a GPER agonist (G-1, 30 µg/kg), or a GPER antagonist (G-15, 300 µg/kg) were administered alone or in combination to young or aged male mice, or young intact or ovariectomized female mice, 1 hour before or 3 hours after cerebral ischemia-reperfusion. Some mice were treated with a combination of G-1 and the pan-caspase inhibitor, quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-VD-OPh), 1 hour before stroke. We evaluated functional and histological end points of stroke outcome up to 72 hours after ischemia-reperfusion. In addition, apoptosis was examined using cleaved caspase-3 immunohistochemistry. RESULTS: Surprisingly, G-1 worsened functional outcomes and increased infarct volume in males poststroke, in association with an increased expression of cleaved caspase-3 in peri-infarct neurons. These effects were blocked by G-15 or Q-VD-OPh. Conversely, G-15 improved functional outcomes and reduced infarct volume after stroke in males, whether given before or after stroke. In contrast to findings in males, G-1 reduced neurological deficit, apoptosis, and infarct volume in ovariectomized females, but had no significant effect in intact females. CONCLUSIONS: Future therapies for acute stroke could exploit the modulation of GPER activity in a sex-specific manner.

Stroke increases g protein-coupled estrogen receptor expression in the brain of male but not female mice.

The novel estrogen receptor, G protein-coupled estrogen receptor (GPER, previously named GPR30), is widely distributed throughout the male and female brain and, thus, could potentially play a role in estrogen-mediated neuroprotective effects in diseases such as stroke. We hypothesized that GPER distribution and expression in the brain of male, intact female, and ovariectomized (OVX) mice is increased after 0.5 h middle cerebral artery occlusion. Using immunohistochemistry, we found that ischemia reperfusion increased GPER distribution in the peri-infarct brain regions of male mice, but surprisingly not in intact females or OVX mice. Similar differences were observed in the male and female human brain after stroke. In contrast, GPER distribution was decreased in the infarct core of all mice examined. Furthermore, GPER immunofluorescence was co-localized with the endothelial cell marker, von Willebrand factor, and the neuronal marker, NeuN. Consistent with the immunohistochemical findings, Western blot analysis showed GPER expression is only elevated in the ischemic hemisphere of male mice. Moreover, GPER mRNA expression in males was elevated at 4 h but had returned to baseline by 24 h. In conclusion, these findings indicate that GPER may be a potential therapeutic target after stroke, especially in males, in whom estrogen therapy is not feasible.

Over-expression of DSCR1 protects against post-ischemic neuronal injury.

BACKGROUND AND PURPOSE: The Down syndrome candidate region 1 (DSCR1) gene is located on human chromosome 21 and its protein is over-expressed in brains of Down syndrome individuals. DSCR1 can modulate the activity of calcineurin, a phosphatase abundant in the brain, but its influence on stroke outcome is not clear. We compared stroke outcome in wildtype (WT) and transgenic (DSCR1-TG) mice which over-express isoform 1 of human DSCR1. METHODS: Transient cerebral ischemia was produced by occlusion of the middle cerebral artery for 0.5 h. After 23.5 h reperfusion, we assessed neurological impairment, brain infarct and edema volume, leukocyte infiltration and markers of inflammation. Intrinsic resistance to apoptosis following glucose deprivation was also assessed in primary cultures of WT and DSCR1-TG neurons. RESULTS: In contrast to WT, DSCR1-TG mice had an improved neurological deficit score, greater grip strength, attenuated infarct volume and brain swelling, and lacked hippocampal lesions after stroke. Expression of mouse DSCR1-1, but not DSCR1-4, mRNA and protein was increased by ischemia in both WT and DSCR1-TG. Brain calcineurin activity was increased to a similar degree after ischemia in each genotype. DSCR1-TG mice had fewer infiltrating neutrophils and activated microglia compared with WT, in association with an attenuated upregulation of several pro-inflammatory genes. Neurons from DSCR1-TG mice were more resistant than WT neurons to apoptotic cell death following 24 h of glucose deprivation. CONCLUSIONS: Over-expression of DSCR1 in mice improves outcome following stroke. Mechanisms underlying this protection may involve calcineurin-independent, anti-inflammatory and anti-apoptotic effects mediated by DSCR1 in neurons.

Brain infarct volume after permanent focal ischemia is not dependent on Nox2 expression.

Reactive oxygen species (ROS) generated by Nox2 oxidase are reported to contribute to infarct damage following cerebral ischemia-reperfusion. Here we have examined for the first time the role of Nox2 expression in outcomes following permanent focal cerebral ischemia. Ischemia was induced by middle cerebral artery filament occlusion (MCAO) for 24h in wild-type (WT) and Nox2(-/y) mice. Neurological deficit and the hanging wire test were assessed, and infarct and edema volumes were estimated using thionin-stained brain sections. Genetic deletion of Nox2 had no effect on any outcome measures at 24h after permanent MCAO. Our data therefore suggest that ROS production by Nox2 oxidase activity plays no significant role in the pathophysiology of cerebral ischemia in the absence of reperfusion.

Neuroprotective effect of an angiotensin receptor type 2 agonist following cerebral ischemia in vitro and in vivo.

BACKGROUND: Intracerebral administration of the angiotensin II type 2 receptor (AT2R) agonist, CGP42112, is neuroprotective in a rat model of ischemic stroke. To explore further its possible cellular target(s) and therapeutic utility, we firstly examined whether CGP42112 may exert direct protective effects on primary neurons following glucose deprivation in vitro. Secondly, we tested whether CGP42112 is effective when administered systemically in a mouse model of cerebral ischemia. METHODS: Primary cortical neurons were cultured from E17 C57Bl6 mouse embryos for 9 d, exposed to glucose deprivation for 24 h alone or with drug treatments, and percent cell survival assessed using trypan blue exclusion. Ischemic stroke was induced in adult male C57Bl6 mice by middle cerebral artery occlusion for 30 min, followed by reperfusion for 23.5 h. Neurological assessment was performed and then mice were euthanized and infarct and edema volume were analysed. RESULTS: During glucose deprivation, CGP42112 (1x10-8 M and 1x10-7 M) reduced cell death by ~30%, an effect that was prevented by the AT2R antagonist, PD123319 (1x10-6 M). Neuroprotection by CGP42112 was lost at a higher concentration (1x10-6 M) but was unmasked by co-application with the AT1R antagonist, candesartan (1x10-7 M). By contrast, Compound 21 (1x10-8 M to 1x10-6 M), a second AT2R agonist, had no effect on neuronal survival. Mice treated with CGP42112 (1 mg/kg i.p.) after cerebral ischemia had improved functional outcomes over vehicle-treated mice as well as reduced total and cortical infarct volumes. CONCLUSIONS: These results indicate that CGP42112 can directly protect neurons from ischemia-like injury in vitro via activation of AT2Rs, an effect opposed by AT1R activation at high concentrations. Furthermore, systemic administration of CGP42112 can reduce functional deficits and infarct volume following cerebral ischemia in vivo.

Importance of T lymphocytes in brain injury, immunodeficiency, and recovery after cerebral ischemia.

Following an ischemic stroke, T lymphocytes become activated, infiltrate the brain, and appear to release cytokines and reactive oxygen species to contribute to early inflammation and brain injury. However, some subsets of T lymphocytes may be beneficial even in the early stages after a stroke, and recent evidence suggests that T lymphocytes can also contribute to the repair and regeneration of the brain at later stages. In the hours to days after stroke, T-lymphocyte numbers are then reduced in the blood and in secondary lymphoid organs as part of a 'stroke-induced immunodeficiency syndrome,' which is mediated by hyperactivity of the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis, resulting in increased risk of infectious complications. Whether or not poststroke T-lymphocyte activation occurs via an antigen-independent process, as opposed to a classical antigen-dependent process, is still controversial. Although considerable recent progress has been made, a better understanding of the roles of the different T-lymphocyte subpopulations and their temporal profile of damage versus repair will help to clarify whether T-lymphocyte targeting may be a viable poststroke therapy for clinical use.

Nox2 oxidase activity accounts for the oxidative stress and vasomotor dysfunction in mouse cerebral arteries following ischemic stroke.

BACKGROUND AND PURPOSE: Post-ischemic oxidative stress and vasomotor dysfunction in cerebral arteries may increase the likelihood of cognitive impairment and secondary stroke. However, the underlying mechanisms of post-stroke vascular abnormalities, as distinct from those causing primary brain injury, are poorly understood. We tested whether augmented superoxide-dependent dysfunction occurs in the mouse cerebral circulation following ischemia-reperfusion, and evaluated the role of Nox2 oxidase. METHODS: Cerebral ischemia was induced in male C57Bl6/J wild-type (WT) and Nox2-deficient (Nox2(-/-)) mice by middle cerebral artery occlusion (MCAO; 0.5 h), followed by reperfusion (23.5 h). Superoxide production by MCA was measured by L-012-enhanced chemiluminescence. Nitric oxide (NO) function was assessed in cannulated and pressurized MCA via the vasoconstrictor response to N(ω)-nitro-L-arginine methyl ester (L-NAME; 100 µmol/L). Expression of Nox2, the nitration marker 3-nitrotyrosine, and leukocyte marker CD45 was assessed in cerebral arteries by Western blotting. RESULTS: Following ischemia-reperfusion, superoxide production was markedly increased in the MCA of WT, but not Nox2(-/-) mice. In WT mice, L-NAME-induced constriction was reduced by ∼50% in ischemic MCA, whereas ischemia-reperfusion had no effect on responses to L-NAME in vessels from Nox2(-/-) mice. In ischemic MCA from WT mice, expression of Nox2 and 3-nitrotyrosine were ∼1.4-fold higher than in the contralateral MCA, or in ischemic or contralateral vessels from Nox2(-/-) mice. Vascular CD45 levels were unchanged by ischemia-reperfusion. CONCLUSIONS: Excessive superoxide production, impaired NO function and nitrosative stress occur in mouse cerebral arteries after ischemia-reperfusion. These abnormalities appear to be exclusively due to increased activity of vascular Nox2 oxidase.

Vascular expression, activity and function of indoleamine 2,3-dioxygenase-1 following cerebral ischaemia-reperfusion in mice.

Indoleamine 2,3-dioxygenases-1 (Ido1) and -2 initiate the kynurenine pathway of tryptophan metabolism. In addition to the established immune regulatory effects of Ido1 and the ability of nitric oxide to regulate Ido1 activity, it is now also known that Ido1-mediated metabolism of tryptophan to kynurenine can modulate vascular tone. Ido activity is reportedly elevated in stroke patients and correlates with increased risk of death. Thus, the present goals were to test whether, following cerebral ischaemia, Ido activity and cerebrovascular Ido1 expression are altered and whether expression of Ido1 contributes to stroke outcome. Transient cerebral ischaemia was induced in wild-type and Ido1 gene-deficient (Ido1 (-/-)) mice. Mice were pre-treated with vehicle, the Ido1 inhibitor, 1-methyl-D-tryptophan (1-MT; 50 mg/kg i.p.) or the inducible nitric oxide synthase (Nos2) inhibitor, aminoguanidine (AG, 100 mg/kg i.p.). At 24 h, neurological function, brain infarct size and swelling were assessed. In addition, Ido activity was estimated by plasma kynurenine and tryptophan, and Ido1 expression was examined in cerebral arterioles. Cerebral ischaemia-reperfusion in wild-type mice increased Ido activity and its expression in cerebral arterioles. Ido1 (-/-) and 1-MT-treated wild-type mice had lower Ido activity but similar post-stroke neurological function and similar total brain infarct volume and swelling, relative to control mice. Inhibition of Nos2 with AG also did not affect Ido activity or outcome following stroke. This study provides molecular and pharmacological evidence that the expression and the activity of Ido1 increase following stroke. However, such Ido1 expression does not appear to affect overall outcome following acute ischaemic stroke, and furthermore, a regulatory role of Nos2-derived nitric oxide on Ido activity following cerebral ischaemia-reperfusion appears unlikely.

Chemokine-related gene expression in the brain following ischemic stroke: no role for CXCR2 in outcome.

This study sought to identify potential targets for acute stroke therapy that can be exploited pharmacologically beyond the current 4.5h time limit for clinical administration of recombinant tissue-plasminogen activator. We used PCR arrays to initially screen the temporal expression profiles of several chemokine-related genes in the brain at 4, 24 and 72h after stroke. We identified large increases (>10-fold) in mRNA at 24 or 72h for the neutrophil CXCR2 receptor, and for CXCL1 and CXCL2-two chemokine ligands expressed by monocytes and neutrophils with strong neutrophil chemoattractant activity via CXCR2. We then tested the efficacy of a CXCR2 antagonist as a therapeutic. Mice were treated with vehicle (1% DMSO) or SB225002 (2mg/kg per day, ip) commencing at reperfusion, and we evaluated chemokine gene expression, neutrophil infiltration and functional and histological endpoints of stroke outcome. Expression levels of CXCL1, CXCL2 and CXCR2 after 24h were markedly reduced to near normal levels in SB225002-treated mice. Myeloperoxidase-positive cell infiltration was significantly reduced in SB225002-treated mice compared with vehicle-treated mice, and was similar to levels in sham-operated mice. However, although SB225002 evidently antagonised the interaction between CXCR2 and its chemokine ligands in the ischemic brain, mice treated with either SB225002 or vehicle had similar motor impairment and infarct volume at 72h. Thus, the reduced expression of CXC chemokine subfamily genes and neutrophil-related infiltration following SB225002 administration did not improve outcome after cerebral ischemia-reperfusion. CXCR2 antagonists are therefore unlikely to be a potential therapy for ischemic stroke.